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Genetic Tools and Techniques

Scientists also use molecular tools to extract and compare bits of a particular kind of RNA from samples in order to determine if previously known or new microbes are present in a particular environment. This technique is widely used as a biomarker and for microbial ecology studies. It uses a particular kind of RNA known as 16S ribosomal RNA, or 16S rRNA.

Ribosomes are the gene-translating machines in all living things. When a gene on a piece of DNA is copied into a strand of messenger RNA and ferried out of the cell nucleus into the cell fluid, ribosomes there latch onto this mRNA. The ribosomes move along the mRNA strand, reading the code contained in its sequence of nucleotide bases (the As, Gs, Cs and Us, since U replaces T in RNA) and stringing the right amino acids together based on the code to build protein chains.

The genes for ribosomal RNA have changed little over millions of years as organisms evolved. The slight changes that have occurred provide clues as to how closely or distantly various organisms are related.

Because the 16S rRNA gene is very short, just 1,542 nucleotide bases, it can be quickly and cheaply copied and sequenced. So when a scientist has a test tube full of pond water or dirt from an arid mountainside, she must first pull out the rRNA that’s mixed up with all the other RNA, DNA and other stuff in that tube. To do this, she cleans and purifies the sample first, getting rid of unwanted debris.

She then uses one or several techniques designed to break open cells like a kid cracking open a piggybank. Now she has to find the 16S rRNA genes in and amongst all the other genes. Although 16S rRNA genes from different microbes will have a few different nucleotides scattered throughout the sequences, those nucleotides at the very beginning or end of the gene are the same from organism to organism.

The scientist uses several copies of another bit of RNA called a primer. A primer is like a mirror image of a short bit of RNA or single strand of DNA; that is, its sequence of nucleotides is the direct complement to the sequence of nucleotides in a known part of the target RNA or DNA.

In this instance, the primer would be the mirror image of the beginning or end of the 16S rRNA sequence. Because complementary nucleotides pair up like the two halves of Velcro, the primer enables the scientist to pick out the 16S rRNA in the sample. The scientist then uses PCR to make millions of copies of these genes. She then has enough 16S rRNA to compare the sequences of the genes from her sample to libraries of stored 16S rRNA genes from numerous known bacteria.

If some of her gene sequences match up perfectly, she knows that these are microbes that have been previously identified. But if others among her sampled sequences show differences, she knows she has found previously unknown microbes.